Hi Anamika,
As far as I understood, the biotin in the elution
buffer is helping your protein to get stripped off from the Avidin column.
So, maybe you dialyze your purified protein (or run FPLC) and get rid of
biotin completely, before you load the protein on to strptavidin coated SPR
chip.
Hope this helps!!
Best,
Bhanu
On Tue, Nov 13, 2018 at 12:14 PM Anamika Singh <[email protected]>
wrote:
> Hi All,
>
> I am purifying the biotinylated protein (cloned into the pET28a vector)
> using Avidin beads. Since I need the protein for SPR but when I used the
> purified protein to interact with Streptavidin coated onto the SPR chip.
> There was no signal. Can anybody tell me why is it so or how can I make
> sure that the purified protein is biotinylated enough to interact and give
> the signal? Because when I ran the SDS-PAGE there was a band of purified
> protein.
>
> I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM
> Biotin).
> PS: I have included the biotin during overexpression of the protein also.
>
> Please suggest.
>
> Thanks
> --
> Anamika
> Post-Doctoral Fellow
> Silberman Institute of Life Sciences
> Hebrew University of Jerusalem, Israel
>
>
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B4U
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